The broad, long-term objective of this proposal is to understand the causes of chronic asthma and thereby improve diagnosis and therapy of this common and debilitating ailment. We showed previously how innate immune activation in response to fungal infection of the airway is linked to the development of T helper 2 (TH2)- biased allergic airway inflammation and associated diseases (asthma and chronic rhinosinusitis). Fungi are ubiquitous in human environments and readily gain access to the airway mucosal membrane through constant inhalation of conidia (spores). We have shown that fungi isolated from the human airway can cause airway hyperreactivity in mice, suggesting that airway fungal growth, i.e., airway mycosis, activates innate and acquired immune responses that could cause asthma in susceptible individuals. This is further supported by our discovery in mice that secreted fungal proteinases cleave fibrinogen in the airways to form cleavage products (FCPs) that activate Toll like receptor 4 (TLR4) to induce fungistatic innate immune responses. However, how these factors mediate allergic inflammation in the lungs remain unknown. Our central hypothesis states that fungal proteinase-mediated cleavage of fibrinogen initiates allergic airway disease and fungistatic innate immune responses in the airways. We will test this hypothesis through the following Aims: 1) Determine the molecular mechanism by which FCPs initiate allergic inflammation and antifungal immunity through TLR4. Hypotheses: Fungal proteinases cleave fibrinogen to yield FCPs that 1) signal through TLR4 via the CD18-CD11b integrin heterodimer (Mac-1) to 2) activate STAT6 and NF-?B. We will use mice with constitutive and targeted deletions of STAT6, NF-?B, and Mac-1 as well as mice harboring a mutation in the fibrinogen gamma chain that prevents binding to Mac-1 to test our hypothesis, confirming our findings using human monocyte derived macrophages. 2) Determine how FCPs initiate allergic inflammation and antifungal immunity through airway epithelia of asthmatics. Hypotheses: 1) Epithelial cells secrete coagulant factors in response to fungal proteinases 2) FCPs initiate allergic and anti-fungal responses mediated by enhanced secretion of airway coagulant factors by airway epithelial cells. We will determine the physiological significance of FCP-mediated induction of clotting factors (e.g., fibrinogen, prothrombin) regarding antifungal immunity and chronic allergic inflammation using animal models of asthma and human airway epithelial cells. 3) Determine the mechanism of innate antifungal immune dysfunction in asthmatics with airway mycosis. Hypothesis: Immune cells from a subset of patients with moderate to severe asthma and airway mycosis are unable to restrain fungal growth in vitro. We will examine the fungistatic ability of human monocyte-derived macrophages (HMDM) against fungal conidia. To resolve the signatures of effective and ineffective innate immune responses to fungi in asthmatics, we will study extreme phenotypes, performing differential transcriptome analyses of HMDM in response to fungal conidia by RNA sequencing.
Rising rates of asthma prevalence and severity in the United States is likely linked to an imperfect understanding of what causes asthma and therapies that are based on this lack of information. Our research has revealed both the potential role of fungi as causes of asthma and how they induce disease through proteases acting on blood clotting proteins. This proposal will extend these findings to develop new and more effective approaches to treating asthma.
